CH654210A5 - PROCEDURE TO GET PREPARED metabolically INCOME OBTAINED FROM YEAST OF ANY KIND. - Google Patents

Abstract

A process for obtaining a sterile, apyrogenic product for promoting oxidative phosphorylation and suitable for therapeutic or cosmetic compositions, starting from yeast, in which any type of yeast is subjected to a process of plasmolysis, followed by treatment with proteolytic enzymes and then with diamine oxidase, after which the proteins present in the solution are precipitated by alcohols, the solution pH is stabilized, and the solution concentrated at low temperature under vacuum.

Description

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1. Method for obtaining a sterile, pyrogen-free, histamine-free, metabolically active product, in particular promoting oxidative phosphorylation, starting from yeast of any type, characterized in that the yeast is subjected in the form of an aqueous dispersion, to a process of plasmosis and homogenization at a temperature below 0 ° C, after a treatment with proteolytic enzymes, then with diaminooxidase is freed from the substances of the histamine type, then by adding a mixture of mono and polyvalent alcohols the fractional precipitation from the solution of the protein substances still present, then the filtered or centrifuged solution is stabilized with buffer solutions at a pH from 6 to 7 and finally concentrated at low temperature, under vacuum, until it has a nitrogen content of at least 1, 4 mg / ml.

2. Process according to claim 1, characterized in that in the fractional precipitation a mixture of alcohols is used comprising:

a) a monovalent aliphatic alcohol with 2-6 C atoms;

b) two or more polyvalent alcohols, possibly partially esterified, containing at least 3 C atoms without: counting the ester groups that may be present, said polyvalent alcohols being in an overall quantity equal to 0.5 * 70-30% by weight with respect to the alcohol monovalent aliphatic;

c) one or more arylaliphatic alcohols with 7-12 C atoms, in a total amount corresponding to 0.05% -0.5% with respect to the monovalent aliphatic alcohol.

3. Process according to claim 2, characterized in that the polyvalent alcohols are selected from: glycerin, sorbite, mannite, inositis, pentaerythrit bis-acetate.

4. Process according to claim 1, characterized in that the aqueous suspension of the starting yeast is added with a bacteriostatic agent compatible with the uses of the final product.

5. Process according to claim 4, characterized in that the bacteriostatic agent is a para-hydroxyalkyl-benzoate.

6. Product obtained according to the process of claim 1, useful as an active principle in the preparation of compositions with therapeutic or cosmetic activity.

The oxidative phosphorylation process is of fundamental importance for all aspects of cell life in aerobic organisms, since it represents the main source of usable energy.

The phenomenon known as oxidative phosphorylation is essentially a process that takes place in chondriosomes and is linked to the formation of "high energy" ATP phosphatic groups through the release of free energy, and where the transfer of energy starts in stages. by an electron transfer during the respiratory process. The term "high energy" is used here to mean those compounds that have a high group transfer potential (25.1-58.6.KJ mole instead of 12.6-20.9 KJ mole of normal phosphoric esters ).

Extensive literature is available on oxidative phosphorylation inhibitors (decoupling, Pasteur effect, antibiotics) while oxidative phosphorylation activators are not known: only extracts of organs from the blood and liver are known in which, however, the ascertainable effects are very modest and for the more are due to foreign substances which in practice increase the oxygen consumption in respiration but not the production of ATP by the cell.

The object of the present invention is the production of active preparations of the intermediate metabolism, starting from the yeast treated in a suitable way.

In the preparation and treatment of the yeast extract according to the present invention, the fundamental problem is constituted by the separation of the unwanted cell components, since in part they perform an inhibiting action, without the activators, through this treatment having to suffer damage.

Operating in a sterile, pyrogen-free environment is a prerequisite for the process. Complete elimination of proteins, including removal of histamine-like substances, are also an essential part of the preparation process.

The yeast treatment process according to the present invention comprises the following essential operations:

1. Plasmolysis and homogenization at temperatures below 0 ° C.

2. Proteolysis with proteolytic enzymes

3. Treatment with enzymes suitable for splitting histamine-type products, in particular diamonooxidase

4. Treatment with alcohol to eliminate proteins

5. pH regulation

6. Concentration under vacuum.

In phase 1, to obtain plasmolysis, a treatment with water is carried out and is then maintained at a temperature of the order of -20 ° C for a few days.

In phase 2 proteolytic enzymes such as pronase, trypsin and ox pancreas are used to eliminate proteins.

Phase 3 consists in the elimination of histamine-type substances with diaminooxidase at pH 6.5-8.0.

Fractional precipitation with alcohols eliminates the residual proteins (phase 4). For the fractional precipitation a mixture of mono and poly-valent alcohols comprising:

a) a monovalent aliphatic alcohol with 2-6 C atoms;

b) two or more polyvalent alcohols, possibly partially esterified, containing at least 3 C atoms (not counting any ester groups), present overall in an amount equal to 0.5% h-30% by weight with respect to the monovalent aliphatic alcohol and preferably selected from: glycerin, sorbite, mannite, inosite, pentaerythrite acetate;

c) one or more arylaliphatic alcohols with 7-12 C atoms, in a total amount corresponding to 0.05% -0.5% by weight with respect to the monovalent aliphatic alcohol.

The pH adjustment in the solution obtained after removing the insolubilized protein substances in the treatment with alcohols of phase 4, is carried out by means of buffer solutions, at pH 6.5 -s- 7.

Suitable buffer solutions are for example those based on citric, lactic, ascorbic, N-alkylsubstituted hexamines.

The pH stabilized solution is finally concentrated under vacuum at low temperature, of the order of -10 ° C or less, until it has a nitrogen content in the concentrate of at least 1.4 mg / ml (phase 6).

The elimination of proteins and histamine-type substances is particularly important when the final product is used for injectable therapeutic compositions.

It is necessary to add a bacteriostatic agent to the suspension of the starting yeast in a percentage compatible with the final product to be obtained. Any bacteriostatic agent which is compatible with the final destination of the prepared product can be used: in particular, para-hydroxy-alkylbenzoates are suitable.

The active preparations obtained according to the present invention have a very high stability over time which allows their practical and convenient use in many forms and with various methods of application.

These active preparations, as mentioned above, carry out an activation of the metabolic oxidation processes which can be represented by the "respiration factor". It has been found that

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this respiration factor for the active preparations according to the invention assumes values from 2.5 to 4. For comparison it can be considered that this factor in the blood extracts reaches 1.5 and in the placenta extracts 1.2.

The determination of the respiration factor of the products prepared according to the invention was carried out according to the Warburg method described by Kleinzeller A. in "Manometrische Methoden" VEB G. FRISCH Verlag, Jena 1965.

The products prepared according to the invention, in the form of a solution, metabolically active, are practically free of toxicity; the DL in the mouse is in fact higher than 20 g / kg.

The observation of the treated mice, in a time interval from 1 minute to 24 hours, shows that all the reflexes, the tone, the coordination -, the emotion, the reactivity, the reflexes connected to the central nervous system are not altered at all compared to the normal state.

The products in question do not cause skin irritation with the tests normally used according to the FD.A's Appraisal of the safety .of chemicals in foods, drugs and cosmetics.

Skin toxicity and eye irritability were determined according to Draize.

The absence of histamine products is confirmed with the cat's text [U.S.P. Pharmacopea XX - pag. 890 (101)].

The administration of the product can take place by both intramuscular and endoperitoneal injection, either intravenously, orally or even topically.

Intravenous administration favorably affects circulatory disorders in particular on peripheral, overall and local circulation; it is particularly useful in acute or chronic ischemias.

Parenteral administration is particularly useful in the regeneration of mucous membranes: for example in the case of ulcers and burns.

Application of the product accelerates tissue formation in plastic surgery.

This effect is expressed on all types of tissue, including bone ones.

It also suppresses tissue irritation due to radiation treatment for therapeutic purposes.

The product according to the invention improves the structure of the skin and therefore can find useful use in cosmetics.

In the fundamental layer of the epidermis, new cells are continuously formed which move those previously formed and push them outwards to the surface of the skin (flaking).

In normal skin, the epidermis is renewed within a month and from the fundamental layer, where it is in contact with the dermis for the intake of nourishment, it moves through the "compact" layer towards the "loose" layer where segregation occurs. In this process, the skin cell passes through the cell stages of the granular layer, cell 5 of the spinous layer and finally the cell of the stratum corneum (keratin) which is flattened, dried and dead. It has now been found that by applying the new product with a vigorous massage on the skin at a concentration of 4% -5% in a suitable ointment, a greater number of new cells are formed by the dermis with the separation of the "disjointed layer" »In the fundamental layer, which cells are characterized by the fact that they are particularly hydrated, rich in cytoplasmic liquid and arranged tightly next to each other. The product is therefore very useful for cosmetic applications.

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Example

Preparation of the metabolically active yeast extract. As starting material, yeast of the Sac-charomyces species is used, for example that of beer or that for bread-making. The yeast is mixed with 5 parts of distilled water containing 0.1% by weight of methyl-para-hydroxybenzoate, and kept at -20 ° C for more than 8 days. After this time it is homogenized at -20 ° C and the pH is brought back to the starting value; then it undergoes the action of proteolytic enzymes at 36.5 ° C. Si 25 uses trypsin and ox pancreas, is maintained at pH 8 and at the above temperature for a minimum of 70 hours and a maximum of 120 hours. It separates with conventional methods, for example by filtration with filter beds or preferably by centrifugation, the unreacted proteins and cellular residues. At this point, a solution is obtained. To eliminate histamine-type substances from the latter, it is treated with diamino-oxidase at pH 6.2-8.0 until the histamine compounds disappear.

Then the solution is subjected to a fractional precipitation by adding a mixture of alcohols to eliminate 35 the residual proteins.

Said mixture consists of: ethanol 10 parts by weight, glycerin 0.2 parts by weight, phenyl-propyl alcohol 0.001 parts by weight, benzyl alcohol 0.02 parts by weight, pentaerythrite acetate 0.0005 parts by weight.

40 It is filtered, or centrifuged, at the end of the precipitation, and the solution is brought to pH 7 with a buffer based on citric or ascorbic or lactic acid and N-alkylglucamine.

The concentration is then continued at -15 ° C and under vacuum of 133-2666 Pa (1 20 mm Hg) until a nitrogen concentration of 1.4 mg / ml is reached in the final preparation.

The latter presents itself as a stable solution over time that has a respiration factor of at least 3.5-4.

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